CN109195971B - Antimicrobial compounds - Google Patents

Abstract

The present invention provides a compound, a process for its preparation and its use as an antimicrobial agent, as well as pharmaceutical compositions comprising it. Also disclosed is the use of actinomycete bacteria to produce the compound (formula III).

Description

Antimicrobial compounds

Technical Field

The present invention provides a compound, a process for preparing the same and its use as an antimicrobial agent.

Background

Natural products remain the most effective source of new antimicrobial drugs, and the chemical diversity of natural compounds remains incomparable with combinatorial chemistry approaches (Newman and Cragg, 2012). Although the latter has been successfully applied to lead optimization, it essentially fails to provide truly new pharmacophores, particularly in the field of antimicrobial drugs. This is mainly due to the limitation of structural variations of the compounds represented by the combinatorial libraries. The majority of the antibiotics in clinical use today are developed from compounds isolated from bacteria and fungi, of which members of the actinomycetes are the main source (Pel a ezF, 2006). The actinomycete-derived antibiotics important in medicine include aminoglycosides, anthracyclines, chloramphenicol, macrolides, tetracyclines, and the like. Traditionally, most of these antimicrobial drugs have been isolated from soil-derived actinomycetes of the genus streptomyces.

However, recent separation strategies have been directed to undeveloped environments, such as marine resources. The focus of biological exploration work has been the isolation and screening of actinomycetes from marine habitats, which has added new biodiversity to the order Actinomycetales and has revealed a range of new natural products with potential pharmacological value (Mincer 2001). The existence of marine actinomycete species that are physiologically and phylogenetically distinct from their terrestrial close-related groups has been widely accepted today, and new marine actinomycete taxa of at least six different families of actinomycete have been described (Fenical et al, 2006).

In addition to phylogenetically differing from their terrestrial close-related groups, marine isolates have been shown to have specific physiological adaptations to their marine environment (e.g., high salinity/osmolarity and pressure). The great diversity of this habitat and its inadequate exploitation are the fundamental reasons attracting researchers to approach it to explore new metabolite producers. Very rare genera appear in marine ecosystems, and many are found to produce new chemically distinct secondary metabolites (Riedlinger 2004), (Zotchev, 2012), (Manivasagan et al, 2014).

Most streptomyces and other filamentous actinomycetes have many gene clusters for the biosynthesis of secondary metabolites (Bentley et al, 2002), and genomic sequence studies have shown that most of their genomes are devoted to the biosynthesis of secondary metabolites. Various gene clusters encoding known or predicted secondary metabolites have been identified in the genomes of different Streptomyces strains (Brautaset et al, 2003) and the marine actinomycete Salinispora (Bode et al, 2002). Many medically important natural products, including antimicrobial and antifungal drugs, are synthesized by these multi-module assembly lines, and genomic mining of secondary metabolite gene clusters has become a common tool for assessing the genetic ability of bacteria to produce new bioactive compounds (Fischbach and Walsh, 2006).

However, even for well-studied model antibiotic producers (such as Streptomyces coelicolor A3(2)), the differences between the number of known metabolites on the one hand and the number of pathways determined from genomic data on the other hand are large (Bentley et al, 2002). These differences can only be explained by the fact that: most gene clusters of secondary metabolites are silent under standard laboratory culture conditions, however expression or upregulation of these pathways is only triggered in response to certain environmental signals. It has been shown that by culturing bacteria under a range of conditions, products of many of these "rare" (orphan) biosynthetic pathways can be obtained (Bode, 2002).

In Engelhardt et al (2010), 27 actinomycetes derived from marine sediments and sponges were classified at the genus level using molecular classification. As described above, PCR screening was performed using genes involved in the antibiotic synthesis of polyketides and non-ribosomal peptides to analyze actinomycetes having the potential to produce biologically active secondary metabolites.

Most antibiotics currently in clinical use were discovered more than fifty years ago. During the past decade, only two new antimicrobial agents with new mechanisms of action have been approved (synthetic oxazolidinone linezolid and the natural product based lipopeptide daptomycin). The loss of efficacy of existing drugs due to the emergence of multidrug resistant pathogens poses a threat beyond the speed of new antimicrobial drug development. Most of all anti-infective drugs are derived from or inspired by natural products. Thus, new antibiotics are most likely derived from natural product-based studies, as neither genomics-derived targets-based studies nor combinatorial chemistry to date provide drugs that actually enter the market.

Therefore, the mining of microbial diversity represents the most promising source for new diverse antimicrobial drugs to address the challenge of emergence of multidrug resistance.

Disclosure of Invention

The present invention provides a compound, and derivatives, salts and solvates thereof, having the structure of formula III

Also provided is a method of producing the compound, comprising the steps of:

a) culturing bacteria selected from the group consisting of:

i) a bacterial isolate deposited under the budapest treaty on 2016 at 7 days 4 months 7 and 4 months by the institute lybnitz DSMZ-german collection of microorganisms GmbH (inhofenstra β e 7B,38124, brelucelix, germany, hereinafter DSMZ) under accession number DSM 32287; and

ii) bacteria closely related to the bacterial isolate of i), such as strains having similar genotypic and/or phenotypic characteristics as the isolated bacteria;

b) extracting the compound of claim 1 from the culture.

In one embodiment of the method of producing a compound, the bacterium is a bacterium comprising in its genome 16S rRNA (which provides a sequence having at least 80% identity to the sequence shown in SEQ ID NO 1 by reverse transcription and second strand synthesis).

Further, it is noted that step b) of the method may comprise the steps of centrifuging the cultured bacteria to obtain a cell pellet from which the compound is extracted, and extracting the compound from the cell pellet using dimethyl sulfoxide (DMSO).

In one embodiment, the culture medium is PM6, optionally containing artificial seawater.

Also provided is the use of an isolated bacterium to produce a compound according to claim 1, wherein the bacterium is selected from the group consisting of:

i) a bacterial isolate deposited by DSMZ at 2016, 4, 7 under the budapest treaty with the accession number DSM 32287; and

b) bacteria closely related to the bacterial isolate in a), such as bacteria having similar genotypic and/or phenotypic characteristics as the isolated bacteria.

Also provided is the use of an isolated bacterium, wherein it is specified that the closely related bacterium comprises in its genome 16S rRNA which provides, by reverse transcription and second strand synthesis, a sequence having at least 80% identity to the sequence shown in SEQ ID NO 1.

Another aspect of the invention is a pharmaceutical composition comprising a compound of the invention and one or more pharmaceutically acceptable carriers and/or adjuvants, and a compound for use in therapy, such as an antimicrobial agent, more particularly an antibacterial agent.

Another embodiment of the invention is a method for treating a bacterial infection in a subject comprising administering to said subject a compound or pharmaceutical composition of the invention.

According to another embodiment, the bacterial infection is caused by multi-drug resistant bacteria (e.g., gram-positive and/or gram-negative bacteria multi-drug resistant bacteria).

Another aspect of the invention is a method of killing bacteria or inhibiting the growth of bacteria comprising the step of contacting a compound or pharmaceutical composition of the invention with the bacteria to be killed or inhibited.

The invention also includes the non-medical use of the compounds as antibacterial agents.

Drawings

FIG. 1 shows the MS spectra (middle) and ESI-of the LC-DAD-isoplot (upper spectrum) and ESI + of the active fractions of MP127-ig17 extract fractionated on HPLC (bottom).

FIG. 2 shows the structure of MBL-AB01 with critical NMR and MS data supporting the structure.

Detailed Description

The present invention provides an antimicrobial agent. The inventors have analyzed actinomycete isolates derived from marine sediments, and have thus identified novel bacteria capable of producing antimicrobial secondary metabolites.

By using microwell, shake flask and fermentor cultures, the inventors were able to determine the culture conditions for the production of antibacterial and antifungal compounds. This method resulted in the identification of the antimicrobial compound, MBL-AB 01.

Accordingly, the present invention provides antimicrobial compounds, such as MBL-AB 01.

Compounds such as MBL-AB01 belong to a group of compounds produced by microorganisms and are commonly referred to as secondary metabolites.

By "secondary metabolite" we mean a compound that can be synthesized by a microorganism. They are not essential for basic metabolic processes such as growth and reproduction. The secondary metabolites may have other useful properties, such as anti-cancer and/or anti-microbial activity, such as antifungal and antibacterial activity (Behal, 2000; Bennett and Bentley, 1989).

The structural elucidation of the compound MBL-AB01 has shown that this compound is a novel compound belonging to the xanthone (Xanthon) class of compounds. The molecular formula is shown as formula I:

the molecular formula is as follows: c₂₇H₁₈ClNO₁₀(I)

The general molecular structure of the compound is shown as formula II:

R₁may be a halogen atom selected from chlorine, bromine and iodine, R₂And R₃May be-O-CH₃。R₄May be-COOH, -C (O) OR₁and-C (O) NR₁R₂And R is₁And R₂Independently of each other is hydrogen, C₁-C₄-alkyl radical, C₂-C₄-alkenyl radical, C₂-C₄Alkynyl radicals and benzeneA radical group.

The compounds according to the invention are xanthones of the general structural formula II.

In one embodiment, the invention has the structure shown in formula I and formula III, and derivatives, solvates and/or hydrates thereof.

The previously known compound xanthholin, has the same formula as shown in formula I. However, comparison of the structures shows a significant difference between the Xantholipin molecule and the MBL-AB01 molecule of the invention. The differences are summarized as follows:

the present invention is a compound having a structure according to formula III, and derivatives, solvates and/or hydrates thereof. As provided herein, a derivative is a compound having a structure according to formula II, wherein R₁Is a halogen atom selected from chlorine, bromine and iodine, R₂And R₃is-O-CH₃。R₄is-COOH, -C (O) OR₁and-C (O) NR₁R₂And R is₁And R₂Independently of each other is hydrogen, C₁-C₄-alkyl radical, C₂-C₄-alkenyl radical, C₂-C₄Alkynyl groups and phenyl groups.

The term "solvate" refers to a solid compound having one or more solvent molecules bound to its solid structure. Upon crystallization of the solid compound from the solvent, a solvate may be formed in which one or more solvent molecules become an integral part of the solid crystalline matrix. The compounds of each structural formula described herein may be solvates. Another type of solvate is a hydrate. "hydrate" likewise refers to a solid compound having one or more water molecules that are tightly bound to its solid or crystalline structure at the molecular level. Hydrates are a particular type of solvate. When a compound solidifies or crystallizes in water, a hydrate may form in which one or more water molecules become an integral part of the solid crystalline matrix. The compounds of each structural formula described herein may be hydrates.

The antibacterial compounds according to the invention are produced by actinomycete bacteria, such as strains of the genus Actinolitenococcus. In a particular embodiment, the antibacterial compounds of the invention are produced by culturing the bacterial isolate MP127-ig17 or closely related strains derived from marine sediments.

By "closely related strain" we mean any strain having similar genotypic and/or phenotypic characteristics as the isolated strain. In particular, the phrase encompasses slightly modified strain forms that retain substantially the same functional activity. Thus, for example, the addition, deletion or alteration of some amino acids or nucleotides has very little effect; the effect, if any, on the functional ability to produce a compound according to the invention. Peak et al set forth a definition of the term "closely related strains," which definition may be used herein.

Further, the present invention provides a method for producing an antimicrobial agent (e.g., MBL-AB01), comprising the step of culturing a bacterium of the actinomycete genus (e.g., a strain of the genus actinomycete heteroclitorius). In one embodiment, the compound is produced by a bacterium selected from the group consisting of: i) a bacterial isolate deposited by DSMZ at 2016, 4, 7 under the budapest treaty with the accession number DSM 32287; and ii) bacteria closely related to the bacterial isolate in i), such as bacteria having similar genotypic and/or phenotypic characteristics as the isolated bacteria.

As described herein, a compound-producing bacterium can be a bacterium that comprises in its genome 16S rRNA that provides, by reverse transcription and second strand synthesis, a sequence that is at least 80% identical, e.g., at least 82%, 83%, 85%, 86%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 98.7%, 98.8%, or 98.9%, or 99% identical, to the sequence set forth in SEQ ID NO: 1.

The skilled artisan will appreciate that considerable changes can be introduced into the sequence defined by SEQ ID NO. 1 and subsequences thereof without significant changes in the overall structure, function and properties.

By "phenotypic characteristic" we mean the ability to produce a secondary metabolite according to the invention (i.e. a compound having a molecular structure according to any of formulae II and/or III).

By "genotypic characterization" we mean the characteristic properties of genetic molecules (e.g. nucleic acids and amino acids, such as 16S rRNA molecules), for example referred to as sequence identity. "strains with similar genotypic characteristics" as referred to herein include bacteria containing 16S rRNA which provides a sequence with at least 80% identity, such as at least 82%, 83%, 85%, 86%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 98.7%, 98.8% or 98.9% or 99% identity, to the sequence shown in SEQ ID NO:1 by reverse transcription and second strand synthesis.

The expression "bacterial isolate" is generally used to define a culture of a bacterial strain. The isolate may be purified and isolated by various methods known to those skilled in the art. As used herein, "bacterial isolate" or "bacterial strain" refers to a microorganism that is genotypically and phenotypically unique, and may originate from a colony, which microorganism is typically obtained by blotting a purified sample onto a suitable medium. The expressions isolate, strain and bacterium are used interchangeably.

According to the process of the invention, the bacteria are cultivated in a microbiological culture process using a suitable medium known to the skilled worker. In one embodiment, the culture medium comprises seawater.

"microbial culture" or microbial culture (microbiological culture) is a method of increasing microorganisms by propagating them in a predetermined medium under controlled laboratory conditions. The term "culturing" is used more generally to mean "selectively growing" a particular microorganism (e.g., a bacterium) in a laboratory.

The compounds according to the invention can be obtained from bacterial cultures as described herein.

Accordingly, the present invention provides a process for the production of a compound of formula III and derivatives, solvates and/or hydrates thereof by culturing a bacterium as described herein in a suitable culture medium in a culture process.

In one embodiment, the medium is a commercially available growth medium commonly used for culturing bacteria, such as tryptone soy broth (Oxoid). In another embodiment, the medium is a standard medium, such as Luria-Bertani (LB) medium. Another embodiment is a complex medium designed for the production of secondary metabolites by actinomycetes (as described by PM6, Engelhardt et al, 2010). In another embodiment, the culture medium is supplemented with artificial seawater. In one embodiment, an inoculum culture containing a bacterial strain producing the compound is produced in a flask containing tryptone soy broth and seawater.

The culture comprising the cultured bacteria may optionally be a production culture. The production culture may be inoculated from a seed culture. The production culture may be produced in flasks containing a suitable medium, such as PM6 medium containing artificial seawater.

By "suitable medium" we mean any medium known to the skilled person to be suitable for culturing the bacterium in question. As used herein, the expression "medium" or "fermentation medium" or "cell culture" refers to the nutrient solution used for growth, and refers to all kinds of media used in the context of culturing isolates. Typically, the medium comprises a carbon source (e.g., sugar, starch, flour or yeast extract), a nitrogen source (e.g., flour containing protein and amino acids or ammonium sulfate), and minerals (e.g., inorganic salts).

The medium may be chemically defined, such as MR6(Illing et al, 1989), complex media (such as PM4, PM5 and PM6, Engelhardt et al, 2010) or standard media (such as ISP 2). Other typical examples of media for the production of antibiotic compounds are R2YE (Thompson et al, 1980), R5(Illing et al, 1989) and AMP (Wendt-Pienkowski et al, 2005).

The method of the invention further comprises the step of isolating the antibacterial compound from the culture. Isolation of compounds from the cultured bacteria can be carried out by methods well known to the skilled person.

One way to obtain the compound is to extract the compound from the production culture and/or from the cell pellet collected by centrifugation of the production culture. This can be done by harvesting the dry matter collected by centrifuging the culture. Optionally, the dry matter can be washed with methanol to extract compounds unrelated to the active compound.

The compound can be extracted by suitable solvents known to the skilled person.

In a particular embodiment, the dry matter from the production culture may be collected by centrifugation and optionally fractionated or lysed (by methods familiar to the skilled person, e.g. by freeze-drying the cell pellet).

In addition, the compounds can be extracted by a suitable solvent, such as by DMSO or by addition of trifluoroacetic acid (TFA) to 0.1% DMSO. Other organic solvents (such as alcohols and alkanes) may also be used to extract the compounds. Undissolved material is optionally removed by filtration. In one embodiment, the compound is further isolated by chromatography (e.g., HPLC). One embodiment is separation by HPLC under basic conditions.

To optionally avoid compound degradation, the pH of each fraction may be adjusted, for example by adding a buffer (e.g., ammonium acetate buffer, pH4) to each fraction collector vial prior to fractionation. The active compound in each fraction will be further bound to a solid phase column which is conditioned with an alcohol (e.g. methanol) and optionally acidified with an ammonium acetate buffer (pH 4). After the compound binds to the column, impurities are washed out of the column acidified with an alcohol (e.g., methanol).

The compound is further eluted from the column with an alcohol (e.g. methanol) optionally further added with an ammonium acetate buffer adjusted to pH 8.0. In addition, the method of isolating the compound may include the step of removing the alcohol or other solvent by vacuum centrifugation prior to adding water and freeze drying the compound.

The method for identifying the compounds of the present invention is performed by using High Performance Liquid Chromatography (HPLC), such as HPLC-MS or HPLC-UV and high resolution Mass Spectrometry (MS).

Engelhardt et al (2010) describe the isolation of marine actinomycete bacteria. This study provided molecular classification and phylogenetic analysis of 27 different actinomycetes. In Table 1, a sponge-derived isolate referred to as TSI127-17 is described. Sequence analysis of the 16S rRNA gene showed that TSI127-ig17 has 98.97% gene similarity to Actinoalloteichous hymeniacidis HPA177(GenAccess No. DQ144222). In Engelhart et al (2010), PCR screening of the PKS/NRPS genes was used to investigate the potential of these actinomycete isolates to synthesize secondary metabolites derived from polyketides and non-ribosomal peptides, thus indicating the potential of these actinomycete isolates to synthesize secondary metabolites.

Provided herein is a deposited bacterium (DSM 32287) comprising in its genome a 16S rRNA molecule having the sequence shown in SEQ ID NO 1.

One aspect of the present invention is the use of a novel bacterial isolate of actinomycete bacteria to produce an antimicrobial compound, such as MBL-AB 01.

The present invention therefore relates to the use of a bacterium of the genus Actinolloteichus (Actinalloteichus) for producing a secondary metabolite, such as the antimicrobial compound MBL-AB 01. The bacteria to be employed may be the antimicrobial compound producing strain Actinalloteichus hymeniacidonis.

In a specific embodiment, the bacterium according to this aspect of the invention is a bacterial isolate (DSM 32287, named MP127-ig17), or a closely related strain as defined herein. In another embodiment, the invention relates to the use of a bacterium selected from the group consisting of: i) a bacterial isolate deposited by DSMZ at 2016, 4, 7 under the budapest treaty with the accession number DSM 32287; and ii) bacteria closely related to the bacterial isolate in i), such as bacteria having similar genotypic and/or phenotypic characteristics as the isolated bacteria.

As described herein, a compound-producing bacterium can be a bacterium that comprises in its genome 16S rRNA that provides, by reverse transcription and second strand synthesis, a sequence that is at least 80% identical, e.g., at least 82%, 83%, 85%, 86%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 98.7%, 98.8%, or 98.9%, or 99% identical, to the sequence set forth in SEQ ID NO: 1. The structural and biological characteristics of the antimicrobial compound MBL-AB01 have been characterized. The compounds of the present invention have been shown to be potent antibacterial agents, shown to inhibit the growth of a variety of bacterial strains, including multi-drug resistant bacteria.

Antibacterial activity has been determined by in vitro studies as described in example 5.

As described in example 6, MBL-AB01 has also been shown to be less cytotoxic in vitro than comparable compounds (e.g. xanthholin). Therefore, MBL-AB01 is a very attractive candidate as an antimicrobial agent for use in different pharmaceutical compositions.

Accordingly, the invention also provides the use of a compound of the invention in medical applications, such as therapy. The invention includes a compound of formula I and/or II and/or III, or a pharmaceutically acceptable salt or solvate thereof, for use in therapy, in particular for the treatment of a bacterial infection.

The terms "treating", "treating" and "treatment" include (i) preventing the occurrence of a disease, pathology or medical condition (e.g., prophylaxis); (ii) inhibiting or arresting the development of a disease, pathological or medical condition; (iii) alleviating a disease, pathology, or medical condition; and/or (iv) reducing symptoms associated with the disease, pathology, or medical condition. Thus, the terms "treatment", "treating" and "treating" can be extended to prophylaxis and can include prophylaxis, prevention, reducing, terminating or reversing the progression or severity of the condition or symptom being treated. Thus, the term "treatment" may suitably include medical, therapeutic and/or prophylactic administration.

The terms "inhibit", "inhibiting" and "inhibition" refer to slowing, stopping or reversing the growth or progression of a disease, infection, disorder or cell population. For example, greater than about 20%, 40%, 60%, 80%, 90%, 95%, or 99% inhibition may be present as compared to growth or progression that occurs in the absence of treatment or contact.

The compounds of the invention and their pharmaceutically acceptable salts or solvates may be used alone but are generally administered in the form of a pharmaceutical composition in which the compound/salt/solvate (active ingredient) is in association with a pharmaceutically acceptable adjuvant, diluent or carrier. The present invention provides such pharmaceutical compositions.

The compounds described herein may be used in the preparation of therapeutic pharmaceutical compositions, for example, by combining the compound with a pharmaceutically acceptable diluent, adjuvant or carrier. The compounds may be added to the carrier in the form of salts or solvates. For example, where the compound is sufficiently basic or acidic to form a stable, non-toxic acidic or basic salt, it may be appropriate to administer the compound as a salt. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form physiologically acceptable anions, such as tosylate, mesylate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, alpha-ketoglutarate, and omicrong-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochlorides, halides, sulfates, nitrates, bicarbonates, and carbonates (Berge et al 1997).

Accordingly, the present invention provides a pharmaceutical composition comprising a compound of formula I and/or II and/or III, or a pharmaceutically acceptable salt or solvate thereof. Such compositions are useful for treating various microbial infections.

The invention also relates to compositions comprising the strains, culture broths, media, inocula, extracts, cell pellets, or compounds of formula I and/or II and/or III and salts thereof of the invention, and to their use for preventing unwanted microbial infections, and to corresponding methods comprising treating animals (including humans) infected with microorganisms with an effective amount of the compositions, strains, culture broths, media, inocula, extracts, cell pellets, or compounds of formula I and/or II and/or III and salts or solvates thereof of the invention.

As used herein, "composition" in reference to a product (microbial strain, agent, compound, or formulation) of the present invention refers to a combination of ingredients, where "formulating" is the process of using a formulation (e.g., a recipe, a combination of ingredients to be added) to form a formulation. Such compositions may also be referred to as formulations. The strains, culture broths, media, inocula, extracts, cell masses or compounds of formulae I and/or II and/or III and compositions of the invention, respectively, are suitable as antimicrobial agents or antibiotics.

The invention also comprises a kit comprising an isolated bacterial culture with the accession number DSM32287, a strain, culture broth, culture medium, inoculum, extract, cell pellet or a compound of formula I and/or II and/or III and salts thereof according to the invention. Kits comprising the isolated bacterial cultures, strains, culture broths, extracts, cell-free extracts, media, inocula or compounds of formula I and/or II and/or III and salts thereof of the present invention are useful for treating a broad spectrum of bacterial infections.

The present invention also provides a process for preparing a pharmaceutical composition of the invention comprising mixing a compound of any one of formulas I, II and/or III, or a pharmaceutically acceptable salt or solvate thereof, with a pharmaceutically acceptable diluent, adjuvant or carrier. The skilled person will be able to determine the appropriate pharmaceutical excipients depending on the route of administration.

Compounds are provided for use as medicaments (e.g., antimicrobial agents, more specifically antibacterial agents).

The in vitro antibacterial activity of the invention was determined for a group of bacterial strains. As shown in the examples, the compounds according to the invention are active against multidrug-resistant gram-positive bacteria, including vancomycin-resistant Enterococcus faecium (Enterococcus faecium).

Also provided is the use of a bacterium (e.g., a deposited bacterial isolate or closely related strain) for the production of a compound.

The present invention provides a therapeutic method of treating an infection in a mammal, the method comprising administering to a mammal having an infection an effective amount of a compound or composition described herein. Mammals include primates, humans, rodents, canines, felines, bovines, ovines, equines, porcines, caprines, bovines, and the like. The infection may be a bacterial infection, for example, an infection caused by a bacterium described herein. Also provided is a method of killing or inhibiting the growth of bacteria comprising the step of contacting a compound according to the invention with the bacteria to be killed. The method may additionally comprise the step of contacting the bacteria with a pharmaceutical composition as described herein.

The ability of the compounds of the invention to treat bacterial infections can be determined by using assays well known in the art. For example, the design of treatment regimens, toxicity assessment, data analysis, quantification of cell death, and biological significance of screening using antibacterial drugs are known. In addition, the ability of a compound to treat a bacterial infection or kill or inhibit bacteria can be determined using the assays described herein.

References in the specification to "one embodiment," "an embodiment," etc., indicate that the embodiment described may include a particular aspect, property, structure, portion, or characteristic, but every embodiment may not necessarily include the aspect, property, structure, portion, or characteristic. Furthermore, these phrases may, but do not necessarily, refer to the same embodiment as those mentioned in other portions of the specification. Further, when a particular aspect, property, structure, part, or feature is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or combine such aspect, property, structure, part, or feature in other embodiments, whether or not explicitly described.

The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes a plurality of such compounds, such that compound X includes a plurality of compounds X. It should also be noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to be taken in conjunction with the recitation of claims or the use of a "negative" limitation, to serve as antecedent basis for use of the associated exclusive terminology (e.g., "alone," "only," etc.).

The term "and/or" means any one of, any combination of, or all of the terms associated with the term.

The term "about" can mean a variation of 5%, ± 10%, ± 20% or ± 25% of the specified value. For example, in some embodiments, "about 50%" may include a variation of 45% to 55%.

As understood by those skilled in the art, all numbers, including numbers expressing quantities of ingredients, properties (e.g., molecular weights, reaction conditions, and so forth) are approximations and are to be understood as being optionally modified in all instances by the term "about". These values may vary depending on the desired properties sought to be obtained by those skilled in the art using the teachings described herein. It will also be understood that these values inherently contain variability necessarily resulting from the standard deviation found in their respective test measurements.

Examples

Example 1 isolation of MP127-ig17

Separation and Classification of MP127-ig17

The isolation of bacterial isolates belonging to Actinoalloteichous hymeniacidis (TSI127-17) has been previously described (Engelhardt et al, 2010).

Soon, sponge samples were collected from 60m deep tatera ridges (63 ° 36'53 "N, 10 ° 31' 22" E, tornado hernand Fjord). The homogenized material was spread on different agar media and the isolate (denoted MP127-ig17) was isolated from agar medium IM18, which was prepared from 0.5 Xnatural seawater and 1ml/l vitamin B solution (thiamine-HCl, riboflavin, nicotinic acid, pyridoxine-HCl, inositol, calcium pantothenate, p-aminobenzoic acid each 5mg/l, 2.5mg/l biotin) containing 3g/l crab meal, 2g/l seaweed meal, 20g/l agar, pH 8.0). MP127-ig17 was unable to grow on this medium in the absence of seawater.

Sequencing of 16S rDNA showed 98.97% gene similarity to Actinoallothecus hymeniacidi HPA 177.

DSMZ has been deposited under the Budapest treaty on MP127-ig17 at 2016, 4, 7, with accession number DSM 32287.

Biological Activity screening

Isolates from sponge samples were incubated at 25 ℃ in different production media and the extracts were screened using the agar diffusion assay as described previously (Engelhardt et al, 2010). The isolate designated MP127-ig17 was cultured in PM6 medium (10 g/l of soluble starch; 2g/l of yeast extract; 10g/l of glucose; 10g/l of glycerol; 2.5g/l of corn steep powder; 2.0g/l of peptone; CaCO33.0 g/l) containing 25% 2X artificial seawater. 2 × artificial seawater was prepared as follows: 1.34g/l KCl, 2.72g/l CaCl₂ x 2H₂O，12.58g/l MgSO₄ x 7H₂O，9.32g/l MgCl₂ x 6H₂O, 0.36g/l sodium bicarbonate, pH 7.8. The solution was sterilized by filtration.

The results show that the extract of MP127-ig17 can inhibit the growth of Micrococcus luteus (Micrococcus luteus) ATCC9341, enterococcus faecium CCUG37832, Candida albicans (Candida albicans) ATCC10231 and Candida albicans CCUG 39434.

Example 2: determination of the bioactive Compound MBL-AB01

An inoculum of MP127-ig17 was produced in a 500ml shake flask containing 100ml tryptone soy broth (Oxoid) and 0.5X artificial seawater. The culture was incubated at 30 ℃ for 5 days. Production cultures (3%, volume/volume) were inoculated from the seed cultures. Production was carried out in 500ml shake flasks containing 125ml PM6 medium (containing 0.5X artificial seawater). The culture was incubated at 25 ℃ for 12 days. The cultures were freeze-dried and extracted with DMSO.

The DMSO extracts were fractionated on an Agilent 1100 series High Performance Liquid Chromatography (HPLC) system with a Zorbax Box-RP column (2.1X 50mm, 3.5 μm) connected to a Diode Array Detector (DAD) and fraction collector system. Methanol and 10mM ammonium acetate (pH4) were used as mobile phase and the methanol gradient increased linearly from 10% to 90% for 24 min. For the entire run, fractions were sampled every minute. The fractions were dried in a vacuum centrifuge and re-dissolved in DMSO.

The activity of each fraction was tested in an automated liquid-based bioassay with micrococcus luteus ATCC9341 and enterococcus faecium CCUG37832 as indicator organisms. Each active fraction was analysed to determine the exact mass of the biologically active compound on an Agilent HPLC system with a Zorbax bones-RP column (2.1 x 50mm, 3.5 μm) connected to a DAD and time of flight (TOF) apparatus. 10mM ammonium acetate (pH 7) and acetonitrile were used as mobile phases and electrospray ionization was performed in negative mode.

The UV absorption peak at 395nm of the active fraction correlates with the peak in the LCMS Q-Tof chromatogram, where the molecular weight of MBL-AB01 is consistent with that of the negative and positive electrospray ionization. After the correct molecular structure was obtained, the molecular weight was calculated to be 551.061927. The spectrum is shown in FIG. 1.

Example 3: the active compound MBL-AB01 was characterized.

Isotopic labeling and molecular formula determination

By having¹³C、¹⁵N or¹³C and¹⁵the molecular formula of MBL-AB01 was determined by production in culture of N-labeled compounds. Seeds were produced in a two-stage culture. First, MP127-ig17 was inoculated into TSB broth supplemented with 50% seawater and cultured for 4 days, and then the seeds were re-inoculated into a medium containing¹³C、¹⁵N or¹³C+¹⁵N-labelled (Silantes) E.coli-OD 2 medium supplemented with 50% seawater was cultured for 6 days. The seeds were transferred to a production medium containing the following composition:comprises¹³C、¹⁵N or¹³C+¹⁵N-labelled (Silantes) E.coli-OD 2 medium; 537ml/l unlabelled or¹⁵N-labelled (NH)₄)₂SO₄(ii) a 0.34g/l MgSO₄ x 7H₂O; 0.17g/l CaCO 3; KH of 2.1g/l₂PO₄(ii) a 0.086g/l of unlabelled or¹³C-labeled glucose; TMS1(Olga Sekurova) at 10g/l

Sletta 1999); 1.3ml/l, and cultured for 11 days. Cultures were freeze-dried, extracted with DMSO, and analyzed as described above. Unlabeled in negative mode (M-H),¹³C-labeled,¹⁵N is labeled with¹³C and¹⁵the masses of N-labeled MBL-AB01 were 550.05, 577.05, 551.05, and 578.14, respectively. Thus, is composed of¹³C and¹⁵the increase in atomic mass caused by the N label indicates that MBL-AB01 has 27 carbons and 1 nitrogen.

Determination of molecular formula and structural resolution by FT-ICR

Characterization of MBL-AB01 was performed by direct injection into Bruker Solarix 12T FT ICR MS equipped with ESI sources. MS spectra were recorded in positive ESI mode and negative ESI mode. The most abundant ions in the spectra were isolated and fragmented by CID. Mass calibration was performed externally using NaTFA standards. The samples were diluted with methanol/water.

Bruker Compass data analysis software was used to predict the likely molecular formula composition of the detected ions. All ions are predicted taking into account the presence of C, H, N, O. In addition, other elements (S, Br, Cl, P, etc.) are included in the search, where isotopic patterns indicate the presence of atoms other than C, H, N and O. The initial prediction was made to allow a mass error of 2 ppm. The theoretical isotopic pattern of the proposed molecular formula is compared to the signal in the MS spectrum. HDX analysis was performed by diluting MBL-AB01 samples in d 4-methanol and CID spectra were recorded after 60 min, 120 min and 240 h.

Isotopic patterns indicate the presence of Cl. Determining the molecular formula as C₂₇H₁₈ClNO₁₀This molecular formula is consistent with the results from the fermentation marker experiments and the isotopic distributions observed with LC-QTOF and FT-ICR. Fragmentation experiments showed that the loss of CO from the proposed molecular ions₂And subsequently losing water. HDX analysis showed that there were a maximum of 5 exchangeable protons.

Structural analysis by NMR

The aim was to determine the chemical structure of the molecule identified as active compound in example 2, where the mass was 551.061927Da and the formula C₂₇H₁₈ClNO₁₀. Xantholipin is the only molecule disclosed in the public domain with this formula, but the data clearly show that MBL-AB01 is not the same as Xantholipin.

A vial of 0.7 mg of purified MP127-ig17 was obtained. The solid material was stored at-18 ℃ until NMR experiments were performed.

The samples were dissolved in 120. mu.l of DMSO-d6 in vials. The solution was transferred to 3mm NMR sample PN 027-20-02. The vial was rinsed with an additional 60. mu.l DMSO-d6 and the wash solution was also transferred to an NMR tube. The tube was flushed with nitrogen before the cap was closed. 800MHz spectrometer with TCO cryoprobe(s) ((¹³C inner coil, optimized for carbon detection).

1D ¹The H spectrum showed a very pure sample of MBL-AB 01. The sample was further analyzed by a number of additional NMR spectra (see fig. 2).

All available NMR and MS data support and are consistent with the proposed MBL-AB01 structure (see formula III). Data and publications on the biosynthetic pathway of related compounds (e.g., xanthholin) also suggest that this structure is rational and fully realizable from a general organic chemistry perspective.

Example 4: isolation of active Compounds from bacterial cultures Using 0.5X Artificial seawater

MP127-ig17 was cultured in a batch fermentation in a 3-liter application fermenter containing 1.65-liter of PM6_ MOD3 medium to produce MBL-AB01 (composition of PM6_ MOD3 medium employed: 30g/l of soluble starch; 2g/l of yeast extract; 2.5g/l of corn steep liquor; 2.0g/l of peptone; CaCO33.0 g/l). The fermentation was carried out at 25 ℃ for 8 days with an initial aeration of 0.25vvm (gas volume flow per minute per unit liquid volume) and then reduced to 15vvm for the remainder of the cultivation and agitation aeration. The dissolved oxygen is over 30%. Seed cultures for fermentation were prepared in 500ml baffled shake flasks containing 100ml tryptone soy broth (Oxoid).

Dry matter from PM6_ MOD3 fermentation broth was collected by centrifugation and then freeze dried. The resulting powder was then washed with 50ml of methanol per gram of powder and extracted twice with 5ml of DMSO per gram of powder and 10ml of DMSO per gram of powder, respectively. The two DMSO extracts were mixed and then lyophilized. The dried extract was resuspended in a small amount of DMSO and the undissolved material was removed by filtration.

The extracts were separated on an Agilent HPLC system with a Zorbax eclipse XBD-C18(5 μm, 9.4X 250mm) column connected to a diode array detector and fraction collector. 0.4ml of 25% NH will be added₃20mM ammonium acetate [ A ]/l]And methanol as the mobile phase. At 76% [ B ]]HPLC was performed in isocratic, initially for 7.5 min. From 7.6 to 9.0 minutes, 100% of B is applied]. The active compound eluted at about 5.5 minutes. To avoid compound degradation, a 0.01 × fraction volume (pH4) of 50g/l ammonium acetate was added to each fraction collector vial prior to fractionation. The active compound in each fraction was bound to a solid phase column (60mg Oasis HLB) adjusted with 100% methanol followed by 76% methanol (pH4) supplemented with 0.1% 50g/l ammonium acetate. After binding the compound to the column, the column was washed with 1.5ml of 85% methanol (pH4), followed by 5ml of 76% methanol (pH 4). The compound was eluted from the column with methanol (pH 8) supplemented with 0.1% 50g/l ammonium acetate. Removing methanol by a vacuum centrifuge; water was added to the compound and freeze-dried.

Example 5: MBL-AB01 in vitro antibacterial activity (MIC assay) compared to other known antimicrobial compounds.

MBL-AB01 was tested against a panel of gram-negative and gram-positive pathogens. The MICs of all gram-positive and gram-negative bacterial strains were determined by standardized microdilution tests using Mueller-Hinton broth (Acumedia). According to the protocols of the clinical and laboratory standards institute, will contain 5X 10 at 35 ℃ in the presence of different antibiotic concentrations⁵The bacterial inoculum of CFU/ml was cultured for 19 hours. Bacterial strains were obtained from culture collection ATCC (american type culture collection), NCTC (national type culture collection) and CCUG (university of goldberg, sweden culture collection).

For most gram-positive strains, the MIC of MBL-AB01 ranged from less than 0.032. mu.g/ml to 0.5. mu.g/ml, comparable to or lower than the MIC of the reference antibiotics vancomycin, gentamicin, streptomycin and daptomycin (Table 1). MBL-AB01 also inhibited the growth of vancomycin-resistant bacterial strains (MICs of 0.25. mu.g/ml and 0.5. mu.g/ml, respectively) represented by enterococcus faecalis CCUG37832 and enterococcus faecium CTC 492.

Enterococcus faecium CCUG 37832: multiple drug resistant, vanA-positive clinical isolates. Mic (μ g/ml): ampicillin (20), chlortetracycline (>10), erythromycin (>20), lincomycin (>10), vancomycin (>20), apramycin (>20), bacitracin (>8), cycloserine (>8), spectinomycin (>8), sensitivity: brevibacterium peptide (0.01)

TABLE 1 in vitro antibacterial Activity of the compound MBL-AB01 against different bacterial strains.

Example 6: determination of in vitro cytotoxicity of MBL-AB01 and Xantholipin on IMR90 fibroblasts

Cytotoxicity of MBL-AB01 and Xantholipin was assessed in vitro using IMR90 human lung fibroblasts (ATCC CCL-186).

MBL-AB01 was isolated as described in example 5 above. Xantholinin is from shanghai university of transportation in china. A stock solution of xantholine was established with methanol and the concentration of the xantholine stock solution was related to the concentration of MBL-AB01 based on UV/vis absorption by assuming that the compound has a similar extinction coefficient at 395 nm.

IMR90 cells were cultured in DMEM-low glucose (Sigma) supplemented with 2mM L-glutamine, 1% MEM NEAA (Sigma), 1mM sodium pyruvate, 10mM HEPES, and 100U/mL Pen-Strep. Cells were subcultured twice or three times per week at a ratio of 1:2 and 1:8 depending on the degree of fusion. 30 μ l of a solution containing 1.2X 10 cells were administered the day before exposing the cells to each compound⁵The cell suspension of IMR90 cells/ml was seeded into 384 well plates (Corning assay plates, 3712) using a Tecan EVO automated workstation with MCA384 pipetting unit and employing a disposable tip (Tecan MCA 125 μ l, 30051808). After inoculation, the plates containing the cell suspension were shaken at 1600rpm and 2.5mm amplitude for 20 seconds (Bioshake). The cell suspension was transferred from the stirred reservoir (reservoir flat bottom, 300mL, 10723363) to the microplate with stirring at 350rpm using a sterile magnetic stir bar (15X 4.5mm VWR 442-4522) on a Tecan EVO. Microplates containing IMR90 cells were incubated at 37 ℃ under an atmosphere of 5% CO 2.

On the day of cell exposure, serial dilutions of MBL-AB01 and xanthholin were made in DMSO. Serial dilutions containing each compound were further diluted in cell culture media and transferred to assay wells provided that the total DMSO concentration in assay wells was 0.6%.

Following exposure, assay plates containing IMR90 cells were further incubated at 37 ℃ under an atmosphere of 5% CO2 for 24 hours. Cell viability after culture was measured using the Promega cell titer-GLO 2.0 viability assay. EC50 values for MBL-AB01 and Xantolipin were estimated based on viability of exposed cells relative to viability of control wells (supplemented with growth medium containing the same DMSO concentration). In this assay, the EC50 value of MBL-AB01 was estimated to be 20 μ g/ml, and the EC50 value of Xanthophyllin against IMR90 cells was estimated to be 1 μ g/ml.

Collection and professional opinion

The applicant claims that the deposited microbial sample (deposited by DSMZ under the Budapest treaty on 2016, 4, 7, and with accession number DSM 32287) can only be used by a professional on the date of patent authorization.

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Claims (14)

1. a compound and salts thereof, the compound having a structure according to formula II:

wherein

R₁Is a halogen atom selected from chlorine, bromine and iodine,

R₂and R₃is-O-CH₃，

R₄is-C (O) OR₅or-C (O) NR₅R₆Wherein R is₅And R₆Independently of each other is hydrogen, C₁-C₄-alkyl radical, C₂-C₄-alkenyl radical, C₂-C₄An alkynyl group or a phenyl group.

2. The compound of claim 1, having a structure according to formula III:

3. a method of producing a compound according to claim 1 or 2, the method comprising the steps of:

a) culturing bacteria selected from the group consisting of:

bacterial isolate, Thermoactinomyces MP127-ig17(Actinalloteichus sp. MP127-ig17), deposited by DSMZ under the Budapest treaty at 2016, 4, 7, with accession number DSM 32287;

b) extracting the compound from the culture.

4. The method according to claim 3, wherein the bacterium comprises 16S rRNA in its genome, the 16S rRNA having the sequence shown in SEQ ID NO 1.

5. The method according to claim 3, wherein step b) comprises centrifuging the cultured bacteria, thereby obtaining a cell pellet, from which the compound is extracted.

6. The method of claim 5, wherein the compound is extracted from the cell pellet with dimethyl sulfoxide (DMSO).

7. The method of claim 3, wherein the culture medium is PM6, optionally containing artificial seawater.

8. Use of an isolated bacterium to produce a compound according to claim 1 or 2, wherein the bacterium is selected from the group consisting of:

the bacterial isolate, Thermoactinomyces MP127-ig17, deposited by DSMZ under the Budapest treaty at 2016, 4, 7, with the accession number DSM 32287.

9. The use according to claim 8, wherein the bacterium comprises 16S rRNA in its genome, the 16S rRNA having the sequence shown in SEQ ID NO 1.

10. A pharmaceutical composition comprising a compound according to claim 1 or 2 and one or more pharmaceutically acceptable carriers and/or adjuvants.

11. Use of a compound according to claim 1 or 2 in the manufacture of a medicament for treating a bacterial infection in a subject.

12. The use of claim 11, wherein the bacterial infection is caused by multiple drug-resistant bacteria.

13. The use of claim 12, wherein the bacterial infection is caused by a gram-positive or gram-negative bacterium.

14. The use of claim 11, wherein the medicament is an antibacterial agent.

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